The invention pertains to the field of precision fluid control for High Performance Liquid Chromatography (“HPLC”) including gradient liquid chromatography (“LC”). Commercially available HPLC systems for carrying out analytical separations typically control fluid at flow rates of a few milliliters per minute (“ml/min”) for columns of 4-5 mm in diameter. The current trend in LC is reducing the size and flow rates of the system in order to reduce the amount of waste generated, lower sample size requirements, and improve compatibility with robust detection systems such as mass spectrometers (“MS”). There is particular interest in columns with diameters in the range of 50 μm to 1 mm that are typically referred to as capillary columns. Since the flow rate of fluid in these capillary systems can range from nanoliters per minute (“nL/min”) to 100 microliters per minute (“μl/min”), there is a need for precision flow control at low flow rates.
The precise control of flow rates is essential to analysis using HPLC. Uncontrolled variations in the flow rate and the fluid composition in HPLC systems produces a number of deleterious effects that compromise the utility and sensitivity of the method. Particularly, a known flow rate is required to reliably predict analyte retention times and thus reliably identify analytes. Also, temporal variations in flow rate, such as pulsations, can produce variations in detector signal that may be confused with the presence of an analyte, resulting in the identification of false analyte features. Temporal variations in flow rate may produce fluctuations in the detector baseline obscuring trace analyte signatures, degrading the minimum detectivity of the system.
The importance of flow control is even more critical for gradient separations where the fluid composition is varied during the course of the separation. In gradient HPLC, the fluid outputs from multiple pumps are summed to provide a desired flow rate of varying composition. Since the retention time of an analyte is very dependent on the time-varying composition of the eluting fluid, precise control of all fluids is critical.
Conventional HPLC pumping systems generally employ positive displacement methods, where the rate of mechanical displacement of a pump element, e.g. a lead-screw driven piston, provides a proportional rate of liquid flow. This method scales down poorly to low flow rates and is unable to control fluid flow with sufficient accuracy to generate reliable and rapid gradients for capillary HPLC systems. The origin of the low flow rate inaccuracies include: check valve leakage, pump seal leakage, flexing and creep of mechanical seals, thermal expansion of components and compression of the working fluid. Many of these issues can produce errors in flow rate larger than the flow rates desired in capillary chromatography. These systems also typically include pulse dampeners and/or fluid volumes to dampen fluctuations due to piston refilling. These volumes produce relatively high hydraulic capacitance in the system. This capacitance, in conjunction with the high hydraulic resistance of microbore columns, leads to slow time response.
The most common approach to achieve flow rates compatible with capillary HPLC is to split the pump output of conventional HPLC pumps. This method is described in Johannes P. C. Vissers, “Recent developments in microcolumn liquid chromatography” Journal of Chromatography A, vol 856 pp. 117-143 (1999); U.S. Pat. No. 6,402,946; and U.S. Pat. No. 6,289,914. In the flow splitting approach, some portion of the mixed solvent is split-off to provide the required flow rate to the column, the balance of the liquid is shunted to waste. The splitting element employs two different flow conductance paths to produce the two flow streams. The precision of this method is limited by changes that may occur in the relative hydraulic conductances of the two flow paths over time. Conductance changes, such as partial plugging of the chromatographic column, will result in changing the flow rate of fluid into the separation column. An additional problem with flow splitting is that it has proven difficult to remove delay times in gradients that result from dead volumes of the splitter based systems. Additionally, these systems generate large volumes of waste relative to the fluid that is actually used since the splitter discards the majority of the fluid in the system.
Agilent Technologies has recently attempted to address one of the problems of flowsplitters using a variable flow splitter with active feedback as disclosed at www.agilent.com. In this capillary HPLC system, high pressure fluids for the gradient separation are delivered by conventional positive displacement pumps and mixed at high volumetric flow rates (˜1 ml/min). The flow rate of the mixture delivered to the column is directly measured and maintained by actively controlling a variable splitter valve. The system is still limited by the delay volumes (˜5 μl) which effects chromatographic performance and the ability to accurately measure flow rates in a varying mixture. At flow rates typical for 300 and 100 μm diameter columns this introduces delay times of 1-10 minutes in the gradient. The delay times make flow rate measurements difficult since they depend on knowing the physical properties of the mixture in the flow sensor at a specific time. Despite the effort to carefully control flow rates, changes in the conductance of the capillary column can result in >20% errors in flow rate and take ˜30 seconds for the system to respond to the conductance change.
In addition, there are several other HPLC systems that have active flow rate control. These systems were developed for high flow rate (>0.1 ml/minute) isocratic HPLC. An early system was developed by DuPont around 1970. The Dupont 833 precision Flow Controller worked together with the Dupont 830 liquid chromatograph (H. M. McNair and C. D. Chandler “High Pressure Liquid Chromatography Equipment-II”, J. of Chrom. Sci., v12, pp425-432 (1974)). The system worked by measuring the pressure drop across a flattened capillary downstream of the pump. The measured flow rate was used to modify the air pressure on a pneumatic piston. The system was designed to work with column diameters of 2.1 to 23.5 mm with high flow rates of up to 100 ml/min.
A gradient system which made use of flow rate feedback and pneumatic actuation was described by Tsukazaki in U.S. Pat. No. 5,777,213. This patent described the advantages for preparative liquid chromatography that typically operates at flow rates of 100's of ml/minute. This system makes use of direct pressurization of a liquid with air which was desirable for medicine or food processing. This method would be very undesirable for capillary HPLC where any bubbles that result from dissolved gasses will dramatically reduce system performance. Additionally, capillary HPLC is typically run at fairly high fluid pressures (greater than 1000 psi) which would make direct pneumatic control a safety concern.
An additional system, disclosed in Jacques C. LeBlanc, “The Stableflow Pump—a low-noise and drift-free pump for high performance liquid chromatography” Rev. Sci. Instrum., v62 pp1642-1646 (1991), was developed for isocratic HPLC using flow rates between 0.1 and 100 ml/minute. This system measures the flow rate after exiting a column and detection cell. The flow rate was controlled by adjusting the temperature of a bath that contained a restricting capillary following the flowmeter. The desired flow rate was achieved using a feedback loop between the flow meter and the temperature bath.
While direct mixing of the fluids is the most robust and useful method for generating gradients, accomplishing this in low flow systems is clearly a challenge.
Accordingly, there is a need in the art for a precision flow control system that is capable of delivering fluid at low flow rates in the range of about 1 nanoliter/minute to about 100 microliters/minute and varying the flow rate in a prescribed manner that is both predictable and reproducible. In addition, it is desirable to have delay volumes of <1 μL and a response time of a few seconds or less.